The long-term objective of the proposed research is to understand the molecular basis of signal dependent membrane protein trafficking in polarized cells. This objective will ultimately require defining the structural features of the sorting signals involved, precise mapping of trafficking pathways in polarized cells, and characterization of the molecular machinery that mediates polarized cell sorting. Knowledge of the molecular basis of membrane protein trafficking in polarized cells such as epithelial cells, endothelial cells and neurons, has potential implications for the transcellular delivery of hydrophilic macromolecular drugs, antigen presentation at mucosal surfaces, as well as the malignant phenotype of some invasive carcinomas. Studies will focus on the trafficking of mutant and chimeric human transferrin receptor molecules expressed in Madin-Darby canine kidney (MDCK) cells using a novel retroviral expression system. The specific aims of this proposal are: 1) to identify and characterize the basolateral sorting signal(s) in the transferrin receptor (TR) cytoplasmic domain; 2) To characterize the trafficking of Ii-TR chimeras in MDCK cells and the sorting signals involved; 3) To identify the structural features of the human TR that determine its apical distribution in polarized brain capillary endothelial cells; 4) To identify dominant-negative mutants of proteins that regulate basolateral sorting and use them to identify the molecular machinery and the intracellular sorting site(s) involved; and, 5) To identify recognition proteins that bind to basolateral sorting signals and to characterize the molecular and kinetic parameters of the interaction by surface plasmon resonance and affinity chromatography techniques. These goals will be accomplished using a combination of molecular biological and electron microscopic methods to analyze the quantitative trafficking of TR molecules in polarized cells. Recent advances in the identification of molecules that regulate membrane protein trafficking in mammalian cells and yeast will be exploited to dissect trafficking pathways in polarized cells. Finally, relatively new techniques will be used to search for molecules that interact with well- characterized basolateral sorting signals